Assessment of Organophosphate Pesticides Exposure in Men with Idiopathic Abnormal Semen Analysis: A Cross-Sectional Pilot Study

Background: Because of the widespread use of organophosphate (OP) pesticides in agriculture, they are major environmental contaminants in developing countries. OP pesticides decrease sperm concentration and affect its quality, viability, and motility. Studies have demonstrated the association between abnormal semen analysis and OP pesticides exposure among the high-risk population. As there is limited data on the percentage of OP pesticides exposure, the study aimed to determine the OP pesticides exposure in Southern Indian men with idiopathic abnormal semen analysis and find the possible source of their OP pesticides exposure. Materials and Methods: In this cross-sectional pilot study, fifty men with idiopathic abnormal semen analysis as cases and fifty men with normal semen analysis as controls were recruited. Detailed history was taken and general and systemic examinations were carried out. OP pesticides exposure was determined by assessment of pseudocholinesterase and acetylcholinesterase levels and urinary OP pesticides metabolites dialkyl phosphate (DAP) consisting of dimethyl phosphate (DMP), diethyl thiophosphate (DETP), and diethyl dithiophosphate (DEDTP). Results: Cases had statistically significantly lower levels of pseudocholinesterase (5792.07 ± 1969.89 vs. 10267.01 ± 3258.58 IU/L) (P=0.006) and acetylcholinesterase [102.90 (45.88-262.74) vs. 570.31 (200.24-975.30) IU/L] (P=0.001) as compared to controls. Cases had a statistically significantly higher percentage of urinary DAP positivity as compared to controls (80 vs. 38%, P<0.0001). Hence, cases had a significantly higher percentage of OP pesticides exposure as compared to controls (20 vs. 4%, P=0.015). OP-exposed cases had significantly higher urinary DETP and DEDTP levels as compared to OP non-exposed cases. Also, urinary DETP and DEDTP levels were significantly negatively associated with sperm concentration, motility, and normal morphology among OP-exposed cases. Conclusion: Southern Indian men with idiopathic abnormal semen analysis had a significantly higher percentage of OP pesticides exposure as compared to men with a normal semen analysis.


Introduction
Infertility as one of the major public health problems is defined as "a failure in achieving a clinical pregnancy after 12 or more months of regular unprotected sexual intercourse" as per the World Health Organization (WHO) (1). According to the WHO, 45-52.6 million married couples were suffering from infertility worldwide in 2010 (2). The prevalence of infertility among Indians was ranging from 3.9 to 16.8% as es timated by the WHO (3). As per the report of a multicentric s tudy by the WHO, 20% of cases of infertility were due to male factors, 38% due to female factors, 27% due to both partners, and 15% cases of infertility were idiopathic. In India, nearly 50% of cases of infertility were due to the reproduction anomaly or disorders in males and in 25% of cases, no detectable causes were found and it was considered idiopathic (4). Male infertility is rising in society and its causes are multifactorial. Many s tudies have shown a declining trend in the semen quality and sperm count among the population (5,6). A s tudy conducted in the Indian population over the pas t 37 years has shown a decline in sperm count and motility, and altered sperm morphology with time (7). No clear cause has been found for the decline of semen quality, but it might be due to environmental, dietary, or other unknown causes (5).
Organophosphate (OP) pes ticides are synthetic chemicals used worldwide for controlling domes tic and agricultural pes ts. Pes ticides used to control pes ts and weed on crops are regis tered under the central insecticides board and regis tration committee (CIBRC), which comes under the Minis try of Agriculture and Farmer welfare. Section-3 of the Insecticides Act, 1968 has regis tered around 30 OP pes ticides, which are in use in India (8). OP pes ticides like monocrotophos, phorate, quinalphos, malathion, chlorpyrifos, diazinon, methyl parathion, ethion, and so on, used extensively in India, were already banned or severely res tricted in the USA and Europe. The OP pes ticides are associated with severe toxicity, contributing to more than 80% of pes ticides-related hospitalization in India (9). OP pes ticides cause phosphorylation of acetylcholines terase resulting in acetylcholine accumulation in synapses. OP pes ticides affect reproduction function by reducing acetylcholines terase activity in the brain, and influencing gonads. OP pes ticides like parathion and methyl parathion have a s tructure similar to hormones like es trogen, thus altering genes expression by interacting with hormone receptors. OP pes ticides alter the hypothalamic-pituitary (HPO), pituitary-thyroid, and pituitary-adrenal axes and serum prolactin levels. OP pes ticides affect spermatogenesis by damaging the Sertoli and Leydig cells and increasing their apoptosis (10). A toxicological s tudy demons trated that OP pes ticides cause low sperm concentration by affecting germ cell proliferation and damaging the seminiferous epithelium (11). Also, OP pes ticides dis turb sperm motility by dis turbing its tail assembly proteins or ATP synthesis (12). Concerning the association between semen parameters and OP pes ticides exposure among agricultural workers, pes ticide sprayers, and workers in pes ticides manufacturing indus tries, several s tudies concluded that there was a decrease in sperm concentration, motility, viability, and normal morphology due to OP pesticides exposure (13)(14)(15)(16)(17)(18). There has been contamination of agricultural soil, sediment, and water by various OP pes ticides throughout India (9). Hence, subtle OP pes ticides exposure is occurring among human beings through food, water, air, tainted breas t milk, playing in the field, or skin contact.
Mos t of the s tudies were done on high-risk populations to find out potential associations between OP pes ticides exposure and alteration in semen parameters. However, there is limited data available in the literature to say that environmental OP pes ticides exposure associates with abnormal semen parameters among the general population. Therefore, the present s tudy aimed to assess the environmental OP pes ticides exposure among Southern Indian men from Pondicherry and surrounding dis tricts of Tamil Nadu, like Tindivanam, Villianur, Chennai, and Villupuram with idiopathic abnormal semen analysis by measuring pseudocholines terase and acetylcholines terase levels and urinary OP pes ticides metabolites. The objec-tives of the s tudy were to compare environmental OP pes ticides exposure between men with and without idiopathic abnormal semen analysis and to determine possible sources of OP pes ticides exposure by comparing percentages of farmers, rural population, smokers, undergraduates, lower socioeconomic s tatus, vegetarians, people using underground water source, and alcoholics.

S tudy design and population
This cross-sectional pilot s tudy was conducted in the Jawaharlal Ins titute of Pos tgraduate Medical Education and Research (JIPMER) hospital, Puducherry, 605 006 from January 2018 to July 2019 after obtaining the approvals from Ins titute Research Council and Ins titute Human Ethics Committee (JIP/IEC/2017/0351 dated November 27, 2017). All Southern Indian men, 25 to 45 years old, from Pondicherry and surrounding dis tricts of Tamil Nadu, like Tindivanam, Villianur, Chennai, and Villupuram, who was attending the JIPMER Infertility Clinic, Pondicherry for the inability of their spouse to conceive after 1 year of unprotected sexual intercourse, were recruited. Written informed consent (both in English and Tamil) after explaining the purpose and the procedure of the s tudy, was obtained from all the participants. After obtaining the consent, detailed his tory of the patient was taken including his occupation, location, source of water, his tory of any medication, or surgery, and other demographic factors. General and sys temic examinations were carried out and the scheduled date for semen analysis was given. On the scheduled day, a semen sample, 10 ml of urine, and 5 ml of blood were collected under s terile conditions. Fifty men who had abnormal semen analysis (sperm count ≤15 million/ml, sperm motility ≤40% and sperm morphology ≤4% normal) as per WHO criteria 2010 (4) with no identifiable pathology, were recruited as cases. Cases with underlying pathology such as varicocele, his tory of diabetes, cardiovascular or thyroid disorders, tuberculosis, tes ticular carcinoma, obs truction or congenital bilateral absence of vas deferens, or use of lipid-lowering drugs, were excluded. Fifty men with normal semen analysis were recruited as controls. So, it was a pilot s tudy with 50 cases and 50 controls, which was accepted by the ins titute research council.

Criteria for analysis of anthropometry, alcohol use, smoking, and socioeconomic s tatus
The height, weight, body mass index (BMI), and wais t circumference were measured by the same observer. The neares t half-kilogram for body weight and half-centimeter for height were recorded. The wais t circumference was determined by measuring the shortes t point below the lowermos t rib cage margin and the iliac cres t and was recorded to the neares t half-centimeter. BMI was calculated as weight (kg) divided by the square of height (m). Being alcoholic was defined by the consumption of at leas t two drinks per day. A s tandard drink was equal to either 10g/12.7 ml of pure alcohol, 330 ml of beer, 100 ml of wine, or 30 ml of s traight spirits or liquor like gin, rum, vodka, or whiskey. Being smoker was defined as having a his tory of smoking over the pas t one year irrespective of the number of cigarettes per day (19). Being vegetarian was defined as eating animal products either never or rarely (less than once per month), consuming dairy products and eggs, but eating meat/ fish less than once per month or who ate fish more than once per month, but other meats less than once per month (20). The socioeconomic s tatus was assessed based on the Kuppuswamy criteria (21).

Semen collection and analysis
Semen was collected in a s terile container by mas turbation in a private room near the laboratory. Participants were asked to abs tain from ejaculation for 3 days before the scheduled date of appointment. Semen volume was measured in a graduated cylinder. Sperm counts were determined on two separate drops of semen using a Neubauer haemocytometer. If the sperm counts determined in the two drops of semen differed by 10% or more, then the count was determined in the third drop of semen. In this case, the sperm count from the firs t two samples which was closes t and within 10% of the third sample count was retained. Sperm count was calculated as the average of the two sperm counts. Sperm motility was determined microscopically in two 10-μl drops from the semen sample. Slides were prepared and observed for altered sperm morphology (22).

Es timation of pseudocholines terase and acetylcholines terase levels
Serum pseudocholines terase levels were measured by sandwich enzyme-linked immunosorbent assay kit from LifeSpan Biosciences, Inc. according to the manufacturer's ins tructions. The intra-assay and inter-assay coefficient of variation (CV) for serum pseudocholines terase was less than 10 and 12%, respectively. Serum acetylcholines terase levels were measured by the Ellman method in which thiocholine, produced by acetylcholines terase, reacted with 5,5-dithiobis (2-nitrobenzoic acid) to form a colorimetric (412 nm) product, proportional to the acetylcholines terase activity present. One unit of acetylcholines terase is the amount of enzyme that catalyzes the production of 1.0 mmole of thiocholine per minute at pH=7.5 at room temperature (23).

Sample preparation and gas chromatography-mass spectrometry
Ten milliliters of urine were collected and s tored at -80°C until further analysis. All urine samples were thawed and mixed by a vortex. Cleaning of urine sample and derivatization of alkyl phosphate were done as per Hemakanthi De Alwis et al. (24). A Trace GC Ultra equipped with AI 3000 Auto-Injector (Rodano, Italy) and ITQ 900 mass spectrometer from Thermo Scientific (Aus tin, USA) was used for analysis with a cons tant flow rate of 1.2 ml/ minutes and Helium as a carrier gas. One microliter of a sample from the low volume insert was injected in splitless mode onto a Thermo Electron Corporation (Rodana, Italy) TR-5MS ([5%-phenyl]-polysilphenylene-siloxane) TRACE GC capillary column (30 m, 0.25 mm, 0.25 μm) using the autosampler. The GCMS protocol followed was as per Hemakanthi De Alwis et al. (24). All the s tandards for dimethyl phosphate (DMP), diethyl thiophosphate (DETP), diethyl dithiophosphate (DEDTP), Sulfotep, an internal s tandard and derivatization agent 2,3,4,5,6-Pentafluorobenzyl bromide were purchased from Sigma-Aldrich with a purity of ≥90%. The mass spectra of the pentafluorobenzyl es ters of DMP, DETP, DEDTP, and Sulfotep was determined. The analysis was done on selective ion monitoring (SIM) mode. The retention time (RT), linearity, the limit of detection (LOD), and limit of quantification (LOQ) for DMP, DETP, DEDTP, and Sulfotep were detected. The data obtained were transferred to X-Calibur files and manually evaluated. The peaks of the samples processed were recognized using the RTs and confirmed by comparison with the analyte/Sulfotep ratio for the two ions of the analyte. Results were reported utilizing creatinine adjus tment.

Organophosphate pes ticides exposure criteria
Both the presence of DAP in urine and inhibition of pseudocholines terase were mandatory for the patients to be labeled as OP pes ticides exposed. Patients with DMP, DETP, and/or DEDTP detected in urine above the LOQ labeled as DAP positive. Proudfoot formula was used as a basis for the determination of inhibition of pseudocholines terase. We considered 4621-11500 IU/L as normal level (> 50%), 2311-4620 IU/L as mild inhibition (20-50%), 460-2310 IU/L as moderate inhibition (10-20%) and less than 460 IU/L as severe inhibition (less than 10%) (14).

S tatis tical analysis
The normality of data was assessed by the Kolmogorov-Smirnov tes t. The dis tribution of categorical data such as socio-demographic s tatus, occupation, being vegetarian, being smoker, being alcoholic, and people using underground water, are expressed as percentages.
The continuous data such as semen parameters, pseudocholines terase and acetylcholines terase levels, and OP metabolites in urine are expressed as mean with s tandard deviation or median (interquartile range). Creatinine was analyzed in urine and all OP metabolites values were adjus ted for creatinine. All OP metabolites concentrations were log-transformed for s tatis tical analysis. Descriptive s tatis tics for OP metabolites among exposed and non-exposed included the percent above the LOD, mean and s tandard deviation, geometric mean and s tandard deviation, ranges, and calculation of the 25 th , 75 th , and 90 th percentile. OP metabolites concentration below the LOD was assigned a value equal to the LOD/√2 (25). Binary logis tic regression was done to es timate relative odd of urinary OP metabolites among exposed and non-exposed groups after adjus tment to age, the number of married years, height, weight, wais t circumference, percentage of undergraduates, lower socioeconomic s tatus, vegetarians, primary infertility and alcoholics. Spearman's or Pearson's correlation was assessed between semen parameters and urinary OP metabolites in the OP-exposed group. Normally dis tributed variables were compared using the s tudent's t tes t. Non-parametric parameters were compared by the Kruskal-Wallis H tes t. S tatis tical analyses were done using SPSS 10 software at a significance level of 5% and P<0.05 was considered significant.

Results
General characteris tics were compared between cases and controls ( Table 1). Cases had a significantly higher percentage of farmers as compared to controls (44 vs. 18%, P=0.009). Similarly, cases had a significantly higher percentage of rural population as compared to controls (60 vs. 38%, P=0.045). Contradictorily, a high percentage of smokers was found among controls as compared to cases (28 vs. 10%, P=0.022). However, no significant difference was found between cases and controls in age, the number of married years, height, weight, wais t circumference, percentage of undergraduates, lower socioeconomic s tatus, being vegetarian, using underground water, primary infertility, and being alcoholic. .001] levels were significantly lower among 40 cases with urinary DAP positivity as compared to 10 cases with urinary DAP negativity. Out of the 40 cases with urinary DAP positivity, 10 (25%) cases had mild inhibition (4417 ± 200 IU/L) and 30 (75%) cases had normal pseudocholines terase levels. Nineteen out of 50 controls had urinary DAP positivity. Out of the 19 controls with urinary DAP positivity, 2 (10.6%) men had mild inhibition and 17 (89.4%) men had normal pseudocholines terase levels. However, all controls with DAP negativity had normal pseudocholines terase levels.
Ten out of 50 cases had both inhibitions of pseudocholines terase and urinary DAP positivity, hence they were labeled as OP pes ticides-exposed. Two out of 50 controls had both inhibitions of pseudocholines terase and urinary DAP positivity, hence they were labeled as OP pes ticides-exposed. Cases had a significantly higher percentage of OP pes ticides exposure in comparison with controls (20 vs. 4%, P=0.015). Also, cases with OP pes ticides exposure had significantly higher urinary DETP and DEDTP levels as compared to cases without OP pes ticides exposure (Table 2). Binary logis tics regression showed that OP-exposed cases had significantly higher urinary DETP (OR=1.12, 95% CI=1.01-1.26), DEDTP (OR=1.27, 95% CI=1.02-1.45) and DAP (OR=1.33, 95% CI=1.13-1.66) levels as compared to non-exposed cases after adjus tment to age, the number of married years, height, weight, wais t circumference, percentage of undergraduates, lower socioeconomic s tatus, vegetarians, people using underground water, primary infertility and alcoholics (Table 3). Correlation analysis among OP-exposed cases showed that urinary DAP levels were significantly negatively associated with sperm concentration (P=0.001, r=-0.634), motility (P=0.001, r=-0.523), and normal morphology (P=0.001, r=-0.721).  To find out the possible source of OP pes ticides exposure, the general characteris tics between OP-exposed cases and non-exposed cases were compared. Percentages of farmers and residing in a rural area were significantly higher in OP-exposed cases as compared to non-exposed cases. However, there was no significant difference in age, BMI, or wais t circumference as well as percentages of men with undergraduate education, lower socioeconomic s tatus, being vegetarian, using underground water, being smokers, and being alcoholics among OP-exposed cases as compared to non-exposed cases.

Discussion
Our s tudy reports that Southern Indian men with idiopathic abnormal semen analysis had a significantly higher percentage of OP pes ticides exposure as compared to men with a normal semen analysis. Also, we found a significant correlation between urinary OP metabolites and semen parameters among OP-exposed cases.
As there is rampant use of OP pes ticides in agriculture, their residues can be found in cooked meals, water, wine, fruit juices, refreshments, and so on. Also, washing and peeling cannot remove the OP residues completely (26,27). Chronic, low-dose exposure to OP pes ticides was found to be associated with neurodevelopmental problems in children, Parkinson's disease, metabolic syndrome, obesity, diabetes, reduced semen quality, reduced ges tational age, reduced birth weight, and so on (28,29). OP pes ticides were found to affect the sperm quality directly or indirectly resulting in infertility and reproduction problems in the agricultural workers. OP pes ticides act as endocrine-disrupting chemicals, alter the HPO axis, and impair spermatogenesis by damaging the Sertoli and Leydig cells (30). The general population is exposed to OP pes ticides mainly through diet, inhalation of air, dermal absorption, and unintentional inges tion (31,32).
Comparing general characteris tics, we noticed that men with idiopathic abnormal semen analysis were mos tly farmers and from the rural area as compared to men with a normal semen analysis. Our observations were consis tent with those reported by Miranda-Contreras et al. (14) who concluded that sperm count, motility, and membrane integrity among Venezuelan farmworkers were affected by occupational pes ticides exposure. Also, Katole and Saoji (33) have reported a lower prevalence of primary infertility among urban populations. Dutta and Bahadur (34) showed that pseudocholines terase and acetylcholines terase levels were decreased among occupationally-exposed tea garden workers of the Northern part of Wes t Bengal, India, similar to our observations. Education has an important role in maintaining personal hygiene, prevention of sexually transmitted disease, and unders tanding the effect of alcohol and smoking on sperm count. Our s tudy has not found any difference between cases and controls in education as the two groups have the same percentage of educated participants.
Many s tudies have used the measurement of urinary DAP as a tool for determining OP pes ticides exposure (13)(14)(15)(16)(17)(18). As Yucra et al. (16) showed that occupation exposure of OP pes ticides cannot be decided solely by OP metabolites measurement in urine, we have included both the determination of DAP in urine and measurement of pseudocholines terase levels for labeling patient as OPexposed. Hence, we may conclude that men with idiopathic abnormal semen analysis had high baseline exposure to OP pes ticides. Li and Kannan (35) es tablished the baseline levels of exposure to OP and pyrethroid pes ticides among the population of several Asian countries. They concluded that India has the second-highes t sum concentration of 11 pes ticides in urine, next to Vietnam. Also, they found that daily intake of chlorpyrifos and parathion was high among the Indian population as compared to the population from other Asian countries. We got higher urinary levels of DEDTP and DETP in cases as compared to controls and these levels were significantly negatively associated with sperm concentrations, motility, and normal morphology. Perry et al. (10) concluded that men with lower semen quality had higher urinary DMP levels as compared to men with normal semen quality. Muñoz-Quezada et al. (36) concluded that urinary DAP levels were high in Chilean school children due to the presence of chlorpyrifos and phosmet residues in fruits.
There is a rising trend of male infertility among the population and for mos t of them, no detectable cause has been found. Hence, it has become the burning ques tion and need of the hour to address what are the possible reasons for the decline in semen parameters? Because there is extensive use of OP pes ticides in agriculture, its contamination in the food chain and its effect on sperm parameters, can sus tain and a low dose of OP pes ticides exposure be one of the causes for the decline of semen parameters among the Southern Indian population? In our s tudy, we found that men with abnormal semen analysis had significantly higher OP pes ticides exposure as compared to men with a normal semen analysis. OP-exposed men were farmers and from the rural population where they might be daily exposed to OP pes ticides through food, water, and air, affecting their sperm parameters.
There were certain limitations in this s tudy: i. We es timated acetylcholines terase activity in serum ins tead of RBC. ii. There were six DAPs: DMP, DMTP, DMDTP, DEP, DETP, and DEDTP. Out of 6 metabolites, we es timated only 3 DAPs i.e. DMP, DETP, DEDTP due to lack of availability of remaining s tandards. iii. His tory of time of recent exposure was not known in our s tudy. Hence, the impact of exposure on the spermatogenesis cycle was not es timated and there was not much information on the chemical insult window period in humans. iv. The seasonal variation of OP pes ticides exposure was not considered in our s tudy. v. Urinary DAP can be derived from pre-formed metabolites in the environment. vi. We have es timated semen analysis on one occasion. We were unable to repeat semen analysis hence characterization was not confirmed. vii. We didn't es timate the hormonal changes in our s tudy population. viii. This observational s tudy has various unmeasured confounders like an ins trumental variable, design, and so on. Due to time cons traints, we have not addressed these confounders.
Though this is a pilot s tudy, it explained a s trong association between unintentional OP exposure and semen parameters. Hence, OP exposure s tatus can be included as one of the inves tigations during the workup of men with an abnormal semen analysis. However, a future s tudy including a larger sample size, more DAP metabolites, collection of more detailed information on demographic and socioeconomic parameters will be required to support our claim.

Conclusion
As OP pes ticides exposure can occur through inhalation, inges tion, and so on, their subtle and chronic exposure is affecting various organs of the human body. The current s tudy showed the effect of OP pes ticides on semen parameters and concluded that men with idiopathic abnormal semen analysis had significantly higher OP pes ticides exposure as compared to men with normal semen analysis. OP-exposed cases had higher urinary OP metabolites levels and more inhibition of pseudocholines terase and acetylcholines terase as compared to non-exposed cases pointing towards a severe degree of OP pes ticides exposure. A higher percentage of OP-exposed men were farmers and from the rural area.